Background: U.S. citizens, particularly military personnel, are vulnerable to the threat of exposure to biological warfare agents. Two such bacterial agents, Bacillus anthracis and Francisella tularensis, can be easily spread by aerosolization causing a high level of mortality, and are therefore considered to be candidate warfare agents. New vaccines against these and other potential warfare agents are needed which can be easily administered and provide high levels of protection against aerosolized bio-weapons. We have developed a Salmonella typhimurium strain (delta-glnA delta-glnH) with a number of attributes that make it an attractive candidate for a live attenuated multivalent vaccine. Our hypothesis is that this attenuated S. typhimurium strain can be used as a single oral vaccine to deliver multivalent antigens and provide both mucosal and systemic protective immunity against aerosolized biological warfare agents, specifically B. anthracis and F. tularensis. We will exploit specific S. typhimurium promoters (e.g., pmrH) to drive high-level expression of B. anthracis and F. tularensis antigens within the lymphoid tissue, and thus generate a sufficient immune response with a single dose. The Specific Aims of this project entail: (1) Construction of delta-glnA delta-glnH attenuated S. typhimurium vaccine strains with the pmrH promoter driving expression of B. anthracis. Protective Antigen (PA) and F. tularensis FopA and TUL4 proteins; (2) Evaluation of the efficacy of vaccine strains (Specific Aim 1) to express heterologous antigens within immune tissue and elicit an appropriate immune response; and (3) Challenge vaccinated animals with aerosolized B. anthracis and F. tularensis to determine efficacy of the vaccine strains. Our Study Design incorporates collaborative vaccine development at three different sites in San Antonio, based upon the expertise found at each site. The S. typhimurium vaccine strains expressing B. anthracis and F. tularensis antigens will be constructed and inoculated into animals in the laboratories of two S. typhimurium researchers, Drs. Karl Klose and John Gunn, at the University of Texas Health Science Center. The evaluation of levels of antigen expression within immune tissue will be carried out at the Brooks Air Force Base by Dr. Kenton Lohman. Aerosolized B. anthracis and F. tularensis challenge studies of vaccinated animals will take place in the Biosafety Level 4 (BSL-4) laboratory at the Southwest Foundation for Biomedical Research under the guidance of Dr. Jean Patterson. We will be taking advantage of this high-level biocontainment laboratory to perform the aerosol challenges necessary to prove the efficacy of this vaccine approach. Relevance: The development of a single oral vaccine that can simultaneously provide protection against multiple bio-warfare agents would be of tremendous benefit to the health of military personnel and other citizens exposed to these agents. If this vaccine strategy proves successful, additional antigens can be expressed from the same vaccine strain, offering an adaptive and protective health tool.